(1). Field of the Invention
The present invention relates to a novel method and kit for proliferation, propagation, maintaining and culturing of eucaryotic cells. In particular the present invention relates to the use of a protein hydrolysate, prepared using one or more hydrolytic enzymes as a basis of a medium for eucaryotic cells.
(2). Description of Related Art
Existing media for eucaryotic cells (culture media) in general comprise mixtures of amino acids, vitamins, carbohydrates and minerals. Culture media contain relatively large amounts of the amino acid L-glutamine. Typically L-glutamine is used in a cell culture medium at a concentration of about 2 mM. L-glutamine is an important energy source in proliferating eucaryotic cells and it also serves as both a carbon and a nitrogen source, especially for purine and pyrimidine synthesis.
The use of L-glutamine as an energy source in cultured mammalian cells proceeds via deamidation of L-glutamine by glutaminase to yield glutamate and ammonia. Glutamate then undergoes transamination to produce .alpha.-ketoglutaric acid which is incorporated into the energy yielding Krebbs tricarboxylic acid cycle.
The incorporation of L-glutamine in a liquid cell culture medium however suffers from the disadvantage that L-glutamine is not stable in the free amino acid form. It is well known to those skilled in the art that it rapidly decomposes into ammonia and pyroglutamic acid. Recently Heeneman et al (J. Immunological methods, 116, 85-91 (1991)) found that as a consequence of this decomposition all tested commercial media contained significantly less L-glutamine than prescribed. In addition, Heeneman et al point to the fact that the formed ammonia can be toxic to cultured cells.
When L-glutamine is incorporated in a peptide it does not decompose. It is stable provided that the L-glutamine residue is not present at the amino terminal side since at this position the L-glutamine residue can decompose into a pyroglutamic acid residue and ammonia. Peptide material containing L-glutamine residues can be obtained via the hydrolysis of suitable proteins or can be prepared synthetically.
There is abundant prior art on the hydrolysis of protein but mainly the use in food products or the use in media to grow micro-organisms is described. In general two types of protein hydrolysates can be distinguished: (1) hydrolysates comprising peptides with a chain length above about 15 amino acids and a relatively low level of free amino acids (below 10%); and (2) hydrolysates comprising peptides with a chain length below 15 amino acids and a relatively high level of free amino acids (about 15% or more).
Hydrolysates from the first group are in food applications mainly used as functional ingredients to aerate or emulsify. It is well known that for optimal functionality peptides with 15-50 amino acids residues are required. The presence of free amino acids should be avoided as these give an unwanted savory taste and smell to the product. Consequently these type of hydrolysates comprise peptides with a chain length well above 15 amino acids and a level of free amino acids below about 10%. To our knowledge hydrolysates from this group do not find an application in media to culture eucaryotic cells.
Hydrolysates from the second group are in food applications mainly used in infant and clinical formulae where a low allergenicity is required. Another preferred characteristic of these products is a reduced bitterness. In both cases the product should contain small peptides and this is achieved via the use of enzyme preparations having both endo- and exo-peptidase activity. As a consequence of the action of the exopeptidases the amount of free amino acids is strongly increased to levels of about 15% or higher.
In the fermentation industries only those hydrolysates from the second group are used which have high amounts of free amino acids (20% and preferably higher). In this case the hydrolysates are used as a relatively cheap source of free amino acids. Protein hydrolysates with a high level of free amino acids however also suffer from the disadvantage that the free glutamine decomposes into pyro-glutamic acid and the toxic ammonia and thus are not very well suited for application in cell culture media.
The prior art (e.g. Animal Cell Culture, A practical approach, second edition, ISBN 0-19-963213-8) describes the use of lactalbumin hydrolysates (prepared with pancreatin which contains both endo- and exopeptidase activity) or other peptones (hydrolysates with a very high level of free amino acids) in cell tissue culture media, but only as supplements and not as the main source of glutamine or other amino acids.
The application of synthetic peptides containing glutamine residues to our knowledge is commercially unattractive due to the very limited availability of such peptides and their relatively high price.
To our knowledge there is no description in the prior art of the use in cell culture media of larger peptides (in the form of protein hydrolysates) or intact proteins containing glutamine residues as the main or only source of glutamine and other amino acids.